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Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA.

Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ - BMC Res Notes (2013)

Bottom Line: The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study.PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured.In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.

ABSTRACT

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

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Correlation between the NF of most three stable and optimal number endogenous control. Pearson’s correlations between the NFs of three endogenous control genes (NF3) and optimal number of endogenous control genes (NFopt) for a) All (for explanation see Figure 4), b) irrespective of stimulation (for explanation see figure 4), c) non-stimulated control, d) LPS stimulated PBMCs, e) LTA stimulated PBMCs, f) LPS and LTA together used for stimulation, and g) PBMCs without culturing (for explanation see Figure 4).
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Figure 6: Correlation between the NF of most three stable and optimal number endogenous control. Pearson’s correlations between the NFs of three endogenous control genes (NF3) and optimal number of endogenous control genes (NFopt) for a) All (for explanation see Figure 4), b) irrespective of stimulation (for explanation see figure 4), c) non-stimulated control, d) LPS stimulated PBMCs, e) LTA stimulated PBMCs, f) LPS and LTA together used for stimulation, and g) PBMCs without culturing (for explanation see Figure 4).

Mentions: The geNorm program calculates the normalization factor assessing the optimal number of reference genes for generating the M factor by calculating the pair-wise variation V. The pair-wise variation between these genes defines the variable V[9]. The lower the variable V is, the less variation. The overall results are shown in Figure 5. Eight endogenous reference genes were required for an accurate normalization factor in the all groups which represents cells in vitro cultured and non cultured together with LPS and/or LTA stimulation (Figure 5a). For the irrespective to stimulation group as shown in Figure 5b, nine endogenous control genes are necessary to obtain the lowest changing V values in the analysed samples. On the other hand, six endogenous reference genes were required for both control and no culture groups (Figure 5c and 5g). For the LPS stimulated group, three reference genes were required to obtain an accurate normalization factor (Figure 5d). For LTA stimulated group, combination four reference genes showed the lowest V value (Figure 5e). However, it is impractical to use excessive numbers of endogenous control genes for normalization, particularly when only a small number of target genes need to be studied or for rare samples that are very difficult to acquire [9,17,30]. Therefore, the use of the three most stable reference genes for the calculation of the NF was considered acceptable for the majority of experiments [9,17,30]. To verify that the use of three reference genes simultaneously is adequate for normalization of RT-qPCR data, the correlation of NF values between the geometric means of the three most stable genes and the optimal number of genes was calculated for each sample groups. As shown in Figure 6, there is a high correlation between the two NF measures (i.e., the theoretical optimal number and proposed number, three) for all groups including irrespective to stimulation group (r = 0.90 to 1, Pearson) (Figure 6a to g). This result demonstrates that the three most stable reference genes are sufficient for an accurate normalization of RT-qPCR data [9,17,30].


Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA.

Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ - BMC Res Notes (2013)

Correlation between the NF of most three stable and optimal number endogenous control. Pearson’s correlations between the NFs of three endogenous control genes (NF3) and optimal number of endogenous control genes (NFopt) for a) All (for explanation see Figure 4), b) irrespective of stimulation (for explanation see figure 4), c) non-stimulated control, d) LPS stimulated PBMCs, e) LTA stimulated PBMCs, f) LPS and LTA together used for stimulation, and g) PBMCs without culturing (for explanation see Figure 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3584940&req=5

Figure 6: Correlation between the NF of most three stable and optimal number endogenous control. Pearson’s correlations between the NFs of three endogenous control genes (NF3) and optimal number of endogenous control genes (NFopt) for a) All (for explanation see Figure 4), b) irrespective of stimulation (for explanation see figure 4), c) non-stimulated control, d) LPS stimulated PBMCs, e) LTA stimulated PBMCs, f) LPS and LTA together used for stimulation, and g) PBMCs without culturing (for explanation see Figure 4).
Mentions: The geNorm program calculates the normalization factor assessing the optimal number of reference genes for generating the M factor by calculating the pair-wise variation V. The pair-wise variation between these genes defines the variable V[9]. The lower the variable V is, the less variation. The overall results are shown in Figure 5. Eight endogenous reference genes were required for an accurate normalization factor in the all groups which represents cells in vitro cultured and non cultured together with LPS and/or LTA stimulation (Figure 5a). For the irrespective to stimulation group as shown in Figure 5b, nine endogenous control genes are necessary to obtain the lowest changing V values in the analysed samples. On the other hand, six endogenous reference genes were required for both control and no culture groups (Figure 5c and 5g). For the LPS stimulated group, three reference genes were required to obtain an accurate normalization factor (Figure 5d). For LTA stimulated group, combination four reference genes showed the lowest V value (Figure 5e). However, it is impractical to use excessive numbers of endogenous control genes for normalization, particularly when only a small number of target genes need to be studied or for rare samples that are very difficult to acquire [9,17,30]. Therefore, the use of the three most stable reference genes for the calculation of the NF was considered acceptable for the majority of experiments [9,17,30]. To verify that the use of three reference genes simultaneously is adequate for normalization of RT-qPCR data, the correlation of NF values between the geometric means of the three most stable genes and the optimal number of genes was calculated for each sample groups. As shown in Figure 6, there is a high correlation between the two NF measures (i.e., the theoretical optimal number and proposed number, three) for all groups including irrespective to stimulation group (r = 0.90 to 1, Pearson) (Figure 6a to g). This result demonstrates that the three most stable reference genes are sufficient for an accurate normalization of RT-qPCR data [9,17,30].

Bottom Line: The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study.PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured.In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Animal Sciences, Unit of Animal Breeding and Husbandry, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.

ABSTRACT

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.

Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

Show MeSH
Related in: MedlinePlus